RSC CC C3CC44016H 3.
Transcript of RSC CC C3CC44016H 3.
6858 Chem. Commun., 2013, 49, 6858--6860 This journal is c The Royal Society of Chemistry 2013
Cite this: Chem. Commun.,2013,49, 6858
Spatially controlled DNA unzipping by microfluidicinterface positioning on a molecule perpendicularto a multicomponent flow†
Naresh K. Mani,abc Sergii Rudiukabc and Damien Baigl*abc
A DNA molecule is rotated under a transversal electric field to be
perpendicular to a longitudinal, multicomponent microfluidic
flow. Positioning the interface allows us to control DNA unzipping
with spatio-temporal resolution. This demonstrates that a micro-
fluidic gradient can be applied at the single molecule level for a
dynamic intramolecular stimulation.
Due to the important roles of molecular gradients in biologicaland synthetic systems, efforts have been devoted to the realiza-tion of controlled gradients at the microscale. The developmentof numerous microfluidic tools1,2 has enabled the creation andfine control of molecular gradients via diffusion-based and/orflow-based approaches.3 Microfluidics-based gradient deviceshave been successfully used to modulate or control variousbiological processes, such as cell migration,4 stem cell differ-entiation,5 axon guidance,6 and embryonic development.7
Microfluidic gradients have also been applied at a single celllevel. For instance, a seminal demonstration was reported byTakayama et al. who succeeded in delivering small moleculesinto selected domains inside an individual cell by a partialtreatment using a laminar multicomponent flow.8 However, toour knowledge, microfluidic gradients have never been appliedto create a controlled perturbation at a single-molecule level. Inthis communication, we describe the application of an electricfield for positioning a single DNA molecule perpendicularly to alongitudinal, multicomponent microfluidic flow in order tocontrol its unzipping, i.e. intramolecular denaturation, withspatio-temporal resolution. The application of electric fields tomanipulate DNA molecules in nano- or microfluidic channelshas been widely used for applications such as DNA stretch-ing,9,10 separation,11–15 concentration,16 mapping17–19 or poly-mer physics investigations.20–25 In contrast to these classical
approaches, where DNA molecules are usually stretched in thesame direction as that of the microfluidic flow, here we appliedthe electric field to rotate a single DNA molecule orthogonallyto a multicomponent microfluidic laminar flow composed of abuffer phase and a denaturant phase.
Fig. 1A shows our experimental set-up. It consisted of amicrofluidic device having two inlets for creating a laminarflow with two components: a DNA buffer solution (upper phase)and a denaturating solution (lower phase). Under our experi-mental conditions, the Peclet number varied between 50 and100 (Table S1, ESI†), which ensured a steep profile of thedenaturating agent concentration around the interface posi-tion. To apply an electric field perpendicular to the flow, twoelectrodes were inserted into reservoirs situated at both ends ofanother channel (the vertical channel in Fig. 1A) perpendicularto the main channel. Finally, a single DNA molecule wastethered to a 10 mm diameter pillar, placed at the crossingregion.
Our strategy for intra-molecular control of DNA unzipping isshown in Fig. 1B. First, in the absence of an electric field anddenaturating phase flow (QD = 0), a double-stranded DNAmolecule (dsDNA) was tethered to the pillar by streptavidin–biotin binding. Then, constant flows of DNA buffer (QB a 0)and denaturing phase (QD a 0) were applied to keep theinterface far from the pillar while stretching the DNA moleculealong the flow (Fig. 1B, top left). Then, the application of anincreasing electric field gradually rotated DNA (Fig. 1B, topmiddle) up to an angle close to 901 when the electrostaticattraction created by the electric field overcame the longitudi-nal force created by the flow (Fig. 1B, top right). The electricfield was then kept constant and the increase in QD allowed usto maintain the interface at different positions, thus inducingpartial DNA denaturation, or unzipping, when a steep denatur-ant concentration gradient was created along the DNA molecule(Fig. 1B, bottom).
The intramolecular unzipping experiment was performed asfollows. First, the PDMS surface of the microfluidic channelwas covalently covered with streptavidin prior to attaching to aPDMS-coated microscopy glass slide (see ESI,† Materials and
a Department of Chemistry, Ecole Normale Superieure, 75005 Paris, France.
E-mail: [email protected]; Web: http://www.baigllab.com/;
Tel: +33 1 4432 2405b Universite Pierre et Marie Curie – Paris 6, 75005 Paris, Francec UMR 8640, CNRS, France
† Electronic supplementary information (ESI) available: Materials and methodsand Table S1. See DOI: 10.1039/c3cc44016h
Received 28th May 2013,Accepted 10th June 2013
DOI: 10.1039/c3cc44016h
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This journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 6858--6860 6859
methods for details). The device was then filled with a 0.3 mMsolution of monobiotinylated lDNA concatemers (3 copies oflDNA per molecule, i.e. 145 500 base pairs, in average) in abuffer composed of 10� Tris-EDTA and YOYO-1 DNA fluores-cent dye (60 nM). At this step, the conjugation led to one orseveral concatemers bound to the pillar and the experimentcontinued only in the former case. After binding of DNA to thepillar, the flow rate of the DNA buffer solution was fixed atQB = 50 nL min�1. Then, the denaturant solution (89% v/vformamide in 10� Tris-EDTA) was added into the microfluidicdevice at a starting flow rate of QD = 5 nL min�1. An electricfield of 400 V cm�1 was applied to rotate DNA. Under theseconditions, DNA could be maintained almost perpendicular tothe flow in the whole range of investigated flow rates. Lowerelectric fields led to smaller angles with respect to the flowdirection while much higher fields induced DNA breakage ordetachment from the pillar.
We applied a stepwise increase in QD (Fig. 2A), and DNAwas observed using fluorescence microscopy. Since YOYO-1is an intercalator that specifically dyes the double-stranded,
non-denaturated part of DNA, we characterized the course oflocal DNA denaturation, or unzipping, by measuring the lengthof the fluorescent dsDNA part (Fig. 2B and C). A first experi-ment was performed at 37 1C, a temperature at which DNA isfully denaturated in an 89% v/v formamide solution. Fig. 2shows that for each denaturant flow rate, dsDNA rapidlyreached a stable length that could be maintained as long asthe flow rate was not changed. For the lower flow rates, a slightdecrease in the time-average dsDNA length was observed (from42.2 � 0.2 mm to 40.4 � 0.4 mm) with an increase in QD from5 nL min�1 to 14 nL min�1. In this regime of QD, the interfaceremained too far from DNA to induce significant denaturation.Interestingly, a strong and stepwise decrease in dsDNA length(17.0 � 0.2 mm and 9.4 � 0.8 mm) was observed with a furtherincrease in QD (17 nL min�1 and 20 nL min�1, respectively).Under these conditions, it was thus possible to maintain asingle DNA molecule in different partially denaturated states,thanks to the local gradient of formamide along the concate-mer. For higher flow rates (QD Z 23 nL min�1), dsDNA couldnot be distinguished anymore, which was attributed to full
Fig. 1 Experimental set-up and concept. (A) A microfluidic device, made ofPDMS, contains two inlets (one for a DNA buffer solution, another one for adenaturant solution), one main channel and two outlets. The main channel iscrossed by another channel, along which an electric field is generated from twoelectrodes. A single pillar (10 mm in diameter) is at the crossing region. Allchannels are 7.5 mm high. (B) A monobiotinylated double-stranded DNA (dsDNA)is attached to the streptavidinated pillar in the absence of an electric field. Theelectric field is increased to bring the DNA molecule perpendicular to the flowaxis (1). Increasing the denaturant flow rate allows one to maintain the interfaceat different positions of the molecule, thus unzipping DNA in a spatio-temporallycontrolled fashion (2).
Fig. 2 DNA unzipping by microfluidic interface positioning. (A) Flow rate of thedenaturant phase (QD) as a function of time. The flow rate of the buffer phase(QB = 50 nL min�1) and the electric field (400 V cm�1) are fixed. (B) Length of adsDNA molecule as a function of time under non-denaturating (T = 25 1C, greentriangles) and denaturating (T = 37 1C, blue circles) conditions. (C) Fluorescencemicroscopy images of the DNA molecule under denaturating conditions as afunction of time. The scale bar is 10 mm.
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denaturation by formamide. To ensure that the extinction ofYOYO-1 fluorescence was due to local denaturation, we per-formed a control experiment at 25 1C, a temperature at whichDNA is not denaturated in a 89% v/v formamide solution.Fig. 2B (green triangles) shows that the whole concatemerremained fluorescent for the whole range of flow rates. Thisshows that DNA can be observed in both formamide and buffersolutions but only when it is double-stranded. Therefore, thedecrease in dsDNA length observed at 37 1C (denaturatingconditions) is unambiguously attributed to the partial DNAunzipping.
We then investigated how DNA unzipping could be spatiallycontrolled by positioning the buffer/denaturant interface. Fig. 3shows the average dsDNA length as a function of QD intriplicate experiments. Although a significant variabilitybetween experiments was observed, three features were repro-ducibly observed. At a low QD, the formamide flow was localizedfar from DNA and no denaturation was observed. Conversely, ata high QD, full denaturation was always achieved. Interestingly,in the intermediate range of QD values, the dsDNA lengthsignificantly decreased with an increase in QD due to thesuccessive positions of the buffer/denaturant interface thatprogressed across the DNA molecule.
We described a microfluidic device in which a single DNAmolecule was electrostatically maintained perpendicular to amulticomponent flow composed of a buffer and a denaturingsolution. We showed for the first time that a simple control ofthe flow rates of the solution allowed us to move the buffer/denaturant interface at different positions across the DNAmolecule, resulting in successive, stable, partially denaturatedstates. The spatial resolution of our approach is of the order ofa few micrometers. Knowing that 1 mm of fully stretched DNAcorresponds to about 3 kbp, which is also the typical size of agene, our method opens the possibility of intramolecularlystimulating large genomic DNA molecules, with a resolutiondown to a single gene level, by using a straightforward androbust hydrodynamic principle. Our concept was demonstratedhere using a buffer/denaturant interface to control a simple
unzipping process. By using other flow compositions, it can beapplied to generate various kinds of chemical or biochemicalstimulations with unprecedented control at the intramolecularlevel. For instance, using compaction agents in one phase willenable dynamic intramolecular control of DNA higher-orderstructure, with immediate applications for synthetic biologyand in vitro gene regulation. Moreover, the use of more elabo-rate gradient-generating devices, by improving the position andsharpness of the interface, will allow intramolecular stimula-tions with improved spatial resolution. Pushing the technologyto a single base-pair resolution, for instance, would create newperspectives for DNA mapping and personal genomics.
This work was supported by the European Research Council(ERC) [European Community’s Seventh Framework Programme(FP7/2007-2013)/ERC Grant agreement no 258782], the InstitutUniversitaire de France (IUF), and the Mairie de Paris[Emergence(s) 2012].
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Fig. 3 Length of the dsDNA molecule as a function of the denaturant flow rate(QD). The flow rate of the buffer phase (QB = 50 nL min�1) and the electric field(400 V cm�1) are fixed. Symbols and error bars show mean values � SD fromtriplicates. Error bars are not shown when they are smaller than the symbol size.T = 37 1C.
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